Cross-Species Metabolomic Analyses in the Brassicaceae Reveals Common Responses to Ultraviolet-B Exposure

Abstract Exposure to UV-B radiation, an intrinsic component of solar light, is detrimental to all living organisms as chromophore units of DNA, RNA and proteins readily absorb high-energy photons. Indirect damage to the same molecules and lipids is mediated by elevated reactive oxygen species (ROS) levels, a side effect of exposure to UV-B stress. To protect themselves from UV-B radiation, plants produce phytochemical sunscreens, among which flavonoids have shown to be particularly effective. The core aglycone of flavonoid molecules is subjected to chemical decoration, such as glycosylation and acylation, further improving sunscreen properties. In particular, acylation, which adds a phenolic ring to flavonoid molecules, enhances the spectral absorption of UV-A and UV-B rays, providing to this class of compounds exceptional shielding power. In this study, we comprehensively analyzed the responses to UV-B radiation in four Brassicaceae species, including Arabidopsis thaliana, Brassica napus, Brassica oleracea, and Brassica rapa. Our study revealed a complete reprogramming of the central metabolic pathway in response to UV-B radiation characterized by increased production of functional precursors of specialized metabolites with UV-B shielding properties, indicating a targeted effort of plant metabolism to provide increased protection. The analysis of specialized metabolites and transcripts revealed the activation of the phenylpropanoid–acetate pathway, leading to the production of specific classes of flavonoids and a cross-species increase in phenylacylated-flavonoid glucosides with synapoyl glycoside decorations. Interestingly, our analysis also revealed that acyltransferase genes of the class of serine carboxypeptidase-like (SCPLs) proteins are costitutively expressed, but downregulated in response to UV-B radiation, possibly independently of the ELONGATED HYPOCOTYL 5 (HY5) signaling pathway.


Introduction
Exposure to high-frequency wavelengths of the solar ultraviolet-B radiation (280-320 nm) has the potential to cause structural modification to DNA, RNA, and proteins as UV-B chromophores present in these molecules readily absorb highenergy photons associated with short-wavelength radiation.Additionally, indirect damage to the same macromolecules and also lipids is caused by the concomitant production of reactive oxygen species (ROS), as these small chemicals containing oxygen become very reactive after accepting high-energy electrons (Halliwell andGutteridge 1985, Jenkins 2009).Therefore, photoautotrophic organisms with obligate dependence on sunlight exposure for the synthesis of carbohydrates have evolved strategies to cope with both the direct and indirect effects of UV-B.At low concentrations, endogenously produced ROS act as cellular signals for cell growth and development (Muhlemann et al. 2018, Chapman et al. 2019).Thus, enzymatic scavengers mainly contribute to ROS quenching and maintenance of ROS cellular homeostasis under standard growth conditions (Waszczak et al. 2018).Conversely, plant-specialized metabolites protect against both direct and indirect UV-B damage associated with either transitory elevated UV-B exposure, for example, in days of high solar radiance, or steady elevated solar radiance typical of lowto-middle latitude and high-altitude zones (Tohge et al. 2016, Chapman et al. 2019).Typically, UV-B protective molecules are constitutively expressed in plants adapted to habitats with high solar irradiation.Nonetheless, specific responses leading to novel molecule synthesis can also be induced upon UV-B exposure, following the activation of the UV RESISTANCE LOCUS 8 (UVR8) UV-B receptor pathway (Rizzini et al. 2011).UVR8 monomers, deriving from the dissociation of UVR8 homodimers after the absorption of high-energy photons, free proteins from proteasomal degradation by interacting with the CON-STITUTIVELY PHOTOMORPHOGENIC 1 (COP1) E3 ubiquitin ligase and concurrently activate the UV-B signaling cascade (Yin et al. 2015, Lau et al. 2019).The basic leucine zipper transcription factor ELONGATED HYPOCOTYL 5 (HY5) is a leading downstream regulator of the UVR8-COP1 response, which, in the dark, is perpetually degraded via COP1 ubiquitin activation (Podolec et al. 2021).HY5 induces the transcriptional regulation of more than 3,000 genes, including genes of the core of the phenylpropanoid pathway as well as enzymes contributing to the decoration of flavonoids produced along the phenylpropanoid-acetate branch (Ulm et al. 2004, Stracke et al. 2010, Rai et al. 2019, Shi and Liu 2021, Job et al. 2022).Plants produce several classes of UV-B protective molecules, and flavonoids constitute one of the most potent groups among all phytochemical sunscreens (Tohge and Fernie 2017).The backbone of a typical flavonoid molecule, which is made of two benzene rings linked by a heterocyclic pyran ring (C6-C3-C6), can absorb high-energy photons and also donate electrons to unstable ROS molecules (Chapman et al. 2019).Hence, flavonoids are structurally built to protect against the direct and indirect effects of UV-B radiation, and in fact, the phenylpropanoidacetate pathway evolved early during the evolutionary history of the green lineage when plants left water and conquered land (Rensing 2018).Flavonoids are grouped into six major classes based on their aglycone moiety, which can be further modified by chemical decorations such as glycosylation and acylation (Tohge et al. 2018).The addition of sugar molecules increases the solubility of the aglycone, for which glycosylated flavonoids can accumulate in higher concentrations inside the vacuoles of epidermal cells.In so doing, they form a superficial layer of phytochemical sunscreen that shields UV-B rays from penetrating deeper inside the tissues (Stracke et al. 2010).Additional decorations, such as the presence of acyl groups, can further increase the UV-B-absorbing properties of flavonoids as demonstrated for the phenylacylated-flavonoid glycoside saiginol A, identified in an LC-MS screen of Arabidopsis thaliana flowers (Tohge et al. 2016).The presence of the additional phenolic ring deriving from the sinapoyl decoration enhances the spectral absorption of UV-A and UV-B rays, providing to saiginols exceptional sunscreen power.The acylation of saiginol A is catalyzed by the flavonol-phenylacyltransferase 2 (FPT2) initially annotated as a serine carboxypeptidase-like (SCPL) protein.In A. thaliana, the FPT2 gene is located on chromosome 2 in a cluster with seven SCPL genes.However, only saiginol-producing accessions with geographical distribution in areas with elevated UV-B radiance carry a functional FPT2 gene, indicating that individual mutations have possibly been filtered out by natural selection in areas with a low solar radiance.While orthologs of the FPT2 gene were only found in Arabidopsis lyrata, cross-species analyses identified two syntenic blocks of SCPL genes in Brassica rapa.
Here, by building on previous discoveries, we expanded the research on phenylacylated-flavonoid glycosides to Brassica napus, B. oleracea, and B. rapa.Our analyses included a comprehensive annotation of primary and specialized metabolites and expression patterns of HY5 and SCPL genes in response to UV-B radiation.

Experimental design
Arabidopsis thaliana plants respond very rapidly to UV-B irradiation.After just a few seconds of exposure to UV-B light, UVR8 homodimers dissociate into monomers, initiating the UV-B signaling cascade (Rizzini et al. 2011).While transcriptional activation is very fast, metabolic responses require longer times to be deployed as they depend on cycles of novel metabolite synthesis and degradation (Kusano et al. 2011).Moreover, the progressive accumulation of metabolites in specific intracellular organelles also requires time, as the process often encompasses the chemical decoration of metabolites occurring in the cytosol, followed by the vesicular transport of compounds into the vacuole (Chanoca et al. 2015).Taking these considerations into account, in this study, we investigated time-point transcriptional and metabolic responses to UV-B light exposure in a comparative experiment between A. thaliana ecotype Col-0 and three species of the Brassicaceae family, namely B. napus, B. oleracea and B. rapa.More specifically, we assessed the responsiveness of each species to promptly activate the transcriptional response to UV-B radiation, which, during the early stages of plant life, is essential for the progression of vegetative development.For this, we assessed the transcriptional responses of genes known to be induced by UV-B exposure in 2-week-old seedlings at seven consecutive time points (0.25, 0.5, 1, 2, 6, 12 and 24 h).Still, whether adult plant can maintain a balanced metabolic state is also relevant for transitioning into the reproductive stage.Therefore, we further assessed changes in primary and specialized metabolite abundance in 4-week-old plants exposed to UV-B light for 2 and 10 h, as well as the abundance of transcripts of key genes in the pathway of flavonoid synthesis at 6 and 8 h.Plant samples harvested immediately before the beginning of the UV-B treatment (time point 0 h) were used as negative controls for all measurements.At the beginning of the UV-B treatment, all seedlings and adult plants have been exposed to 3 h of visible photosynthetic light devoid of UV-B frequencies; hence, they were actively photosynthesizing.CHALCONE ISOMERASE (CHI) was also shown to be mediated by HY5 acting together with zinc-fingers of the BBX family, which provide the transcriptional activation potential (Job et al. 2022, Podolec et al. 2022).The results of a recent analysis of transcripts and genomic sequences suggested that the core UVR8-COP1-HY5 signaling pathway originated in a common ancestor when plants emerged from water and developed common means of protection toward the damaging effect of the solar radiation (Han et al. 2019).To investigate whether the activation response by HY5 is conserved across the Brassicaceae species, we measured the steady-state level of HY5 transcript abundance in 2-week-old seedlings at specific time points of exposure to UV-B radiation (Fig. 1A).In all species, including A. thaliana, HY5 expression peaks 2 h after exposure to UV-B light, which indicates that the activation of the response to UV-B radiation proceeds in all Brassicaceae species with a similar trend.Moreover, despite the accumulation of HY5 transcripts starts to decrease after 2 h of UV-B treatment, it is still significantly elevated after 6 h of exposure or longer (Fig. 1B).Moreover, significant levels of HY5 transcripts are already evident after 1 h of exposure or just 30 min as in B. napus.These results revealed that the pattern of HY5 expression is well conserved across all Brassicaceae species and characterized by a strong and prompt initial induction happening soon after the first exposure to UV-B irradiation.

Transcriptional responses to UV-B exposure in young seedlings and expression of SCPL genes
In A. thaliana, a member of the SCPL family named FPT2 was shown to catalyze the acylation of the recently characterized phenylacylated-flavonoid glycoside saiginol A, which confers increased fitness to ecotypes adapted to areas with high solar radiance (Tohge et al. 2016).The FPT2 gene localizes on chromosome 2 in a tandem duplication with seven SCPL genes (Fig. 2A).The tandem duplicated gene cluster includes SCPL12 (FPT1, At2g22920), pSCPL (FTP2, At2g22960), SCPL11, (At2g22970), SCPL13 (At2g22980), SCPL8 [sinapoylglucose:malate sinapoyltransferase (SMT), At2g22990], SCPL10 [sinapoyl-glucose: anthocyanin sinapoyltransferase (SAT), At2g23000], and SCPL9 [sinapoyl-glucose:sinapoyl-glucose sinapoyltransferase (SST), At2g23010].The seven SCPL genes display high sequence similarity and share a common sinapoyl donor but have specific sinapoyl acceptors.In this study, orthologs of A. thaliana SCPL genes in the Brassicaceae species were identified by comparative genomic analysis using syntenic relationships and utilized to build a phylogenic tree in which the known A. thaliana SCPL genes encoding for SAT, FPT2, SMT and SST enzymes served for the identification of potential functional clades (Fig. 2B).As genes belonging to a same clade are supposed to have a similar enzymatic function, with this method, we identified the putative SCPL genes A04p17550 and A03p27310 in B. napus, Bo4g151230 and Bo3g042330 in B. oleracea, and Brara.I04557 in B. rapa.Therefore, on the same set of samples described earlier, in which overexpression of HY5 displayed UV-B-dependent induction, we measured the level of transcripts of SCPL genes and the orthologs.Conversely to our expectations, the results of these experiments showed that all SCPL genes, irrespective of the species, displayed a progressive downregulation upon UV-B exposure, revealing that SCPL expression is constitutive rather than inducible (Fig. 3).Whether HY5 is responsible for the downregulation of SCPL genes in response to UV-B is intriguing to postulate.However, this hypothesis would be better resolved in hy5 mutant lines of all the Brassicaceae species.Nonetheless, as the pattern of SCPL expression does not follow the pattern of HY5 transcriptional activation, it is possible that the SCPL expression is governed by a different transcriptional activator.
Fig. 2 The A. thaliana SCPL gene cluster and phylogenetic analysis of SCPL genes in the Brassicaceae.(A) A schematic representation of the SCPL gene cluster on chromosome 2 of A. thaliana and the metabolic function of characterized genes in the cluster.(B) The phylogenetic tree of SCPL genes in Brassicaceae.SNG1, sinapoyl-glucose accumulator 1.

Metabolites of the central pathway displayed the accumulation of functional precursors for targeted specialized metabolite synthesis across all Brassicaceae species
As the metabolites of the central pathway provide precursors for the synthesis of specialized metabolites, we utilized a wellestablished gas chromatography-mass spectrometry method (Lisec et al. 2006, Alseekh et al. 2021) to investigate the effect of UV-B radiance on the steady-state accumulation of these compounds.Then, to facilitate data interpretation, peak areas of each specific metabolite measured after 2 and 10 h of exposure were compared to time 0 and plotted on a heatmap of metabolite abundance distributed along the graphical representation of the central pathway (Fig. 4; Supplementary Dataset S1).As it has already been shown that UV-B radiation induces significant metabolic changes in young seedlings (Kusano et al. 2011), we focused our research on adult plants, in which the maintenance of a balanced metabolic state is relevant for the maintenance of plant physiological functions and transitioning into the reproductive stage.Starting from the pathway of glycolysis, our measurements revealed species-specific responses to UV-B exposure as metabolites and carbohydrates immediately related to the pathway differentially accumulated in the four species under study.Indeed, while the content of the three major sugars (glucose, fructose, and sucrose) significantly decreased in Col-0 after only 2 h of exposure, glucose and fructose slightly increased in all the remaining Brassicaceae, with a significant increment for sucrose in B. rapa.After 10 h of treatment, the responses of B. rapa and A. thaliana started to align, while B. napus and B. oleracea were still clustered together.The metabolic responses were more leveled across species and time points for the sugar alcohols glycerol, galactinol, and myoinositol.Nevertheless, A. thaliana displayed a significant reduction in the content of all these metabolites.High raffinose was measured in B. napus and partially in B. oleracea, which may reflect a particular form of carbon mobilization typical of these species.In general, the responses observed for Col-0 align with the relative metabolite abundance recently measured after 24 and 96 h of UV-B treatment, which indicates that the decline in sugar content progressively aggravates over long-time exposure (Job et al. 2022).
The pressure exerted by UV-B light on the intermediates of the tricarboxylic acid (TCA) cycle was also elevated in Col-0, as citrate, 2-oxoglutarate, succinate, and fumarate displayed reduced contents.Cross-species differences leveled off for the content of many amino acids, including the branched amino acids Leu, Val, and Ile, as well as Ser, Lys, Thr, and Asn, which all displayed significant increases over 10 h of UV-B exposure.A similar trend was observed for the aromatic amino acids, of which the content progressively increased over time.Interestingly, despite UV-B exerting differential pressure on specific metabolite nodes based on the species, Phe, the precursor of phenylpropanoid metabolism, uniformly increased in all species.A similar trend was observed for the metabolites of two branches of the Met biosynthetic pathway, which starts from Ser and Asp, and diverts toward Thr and Ile on one branch and Cys and Met on the other.As the biosynthesis of phenolic compounds requires significant methylation, it is compelling to speculate that given the high demand for the cofactor S-adenosylmethionine for methylation reactions, all species equally invest in providing a sufficient number of precursor molecules to face increased methylation demand.Pro, the metabolic marker of stress responses in most plants (Ghosh et al. 2022), only increased significantly in Col-0 and B. oleracea.Finally, it is worth mentioning the slight increase in dehydroascorbate observed in all species except for B. napus.Cellular ROS production is a universal response in organisms exposed to UV-B rays and handled, in the first place, by enzymatic scavengers.Ascorbate peroxidases utilize ascorbate as a reducing agent to detoxify hydrogen peroxide, and in doing so, they release monodehydroascorbate, which spontaneously converts into dehydroascorbate.As the intracellular pool of glutathione is gradually consumed when exposure to UV-B is protracted, less glutathione becomes available to the dehydroascorbate reductase enzyme, for which dehydroascorbate may accumulate in plant tissues.
In conclusion, UV-B light significantly impacted the central metabolic pathway as inferred from changes in metabolite content observed after 2 and 10 h of UV-B exposure.Nonetheless, all Brassicaceae displayed increased production of functional precursors of specialized metabolites with UV-B shielding properties, indicating a targeted effort of plant metabolism to provide increased protection.

Analysis of specialized metabolites and transcripts revealed species-specific production of UV-B-protective compounds and conserved production of sinapoyl glucose
The transcriptional activation of the flavonoid pathway in response to UV-B light has been reported in many species, which highlights the ancestral origin of this response now shared among plants adapted to life on land (Han et al. 2019).Studies mostly performed in knockout mutants of A. thaliana revealed the vital role of flavonoids for plant fitness under UV-B light (Ruegger andChapple 2001, Dean et al. 2014), while screens of natural accessions identified classes of compounds with enhanced sunscreen shielding properties, which are typical of ecotypes adapted to environments with strong solar radiance (Tohge et al. 2016).Therefore, we assessed whether adult plants are also capable of mounting a transcriptional response to UV-B radiation, which ultimately will induce the activation of the pathway of flavonoid synthesis.For this, we harvested the aboveground portion of plants exposed to UV-B light and extracted RNA and metabolites for further analysis.
First, we measured the expression of HY5 and observed that, in all species, the number of transcripts was significantly elevated till 6 h upon UV-B exposure and up to 8 h in B. napus (Fig. 5).Then, we measured the level of transcripts of key genes of the core phenylpropanoid pathway, as well as genes located after the branching point between the phenylpropanoid and the phenylpropanoid-acetate pathways, such as FERULIC ACID 5-HYDROXYLASE (F5H) and COUMARATE 3-HYDROXYLASE (C3H).Upon 6-h exposure to UV-B light, a general activation of the core phenylpropanoid pathway was revealed by the accumulation of transcripts of PHENYLALA-NINE AMMONIA-LYASE (PAL), CINNAMATE 4-HYDROXYLASE (C4H), and 4-COUMARATE:CoA LIGASE (4CL1 and 4CL3) genes.These results align with what we had inferred from the analysis of primary metabolites, where we measured an increased content of Phe, which uniformly appeared in all species under study (Fig. 4).Similarly, transcripts of F5H and C3H genes, which are involved in the biosynthesis of lignin (Alber et al. 2019, Sakamoto et al. 2020), revealed a general increase upon UV-B exposure.
As the results of these measurements revealed a consistent transcriptional activation of the phenylpropanoid pathway, to assess whether transcriptional responses are followed by the increased accumulation of specialized metabolites, we applied a liquid chromatography-mass spectrometry method (Tohge andFernie 2010, Perez de Souza et al. 2021) to separate and annotate compounds and determine their accumulation at a very early (2 h) and late (10 h) time points upon exposure to UV-B light.Our analysis identified a total of 34 compounds in A. thaliana ecotype Col-0, 49 compounds in B. napus, 45 in B. oleracea, and 52 in B. rapa, with 18 compounds shared among all species (Fig. 6; Fig. S1; Supplementary Dataset S2).Thirteen compounds were previously identified by co-elution peaks of leaf extracts from A. thaliana (Tohge et al. 2016), while additional 11 compounds were annotated by the interpretation of specific in-source fragmentation patterns of the aglycone moiety and the corresponding decorated compound.Finally, to facilitate cross-species comparisons, peak areas measured for each compound after 2 and 10 h of UV-B treatment were compared to time 0, plotted on a heatmap of clustered metabolites based on similar patterns of abundance (Fig. S1), and the metabolites revealing the most significant changes were represented as bar plots (Fig. 6).Overall, our analysis revealed a temporal decrease in indolic glucosinolate compounds contrasted by an increase in flavonoids including anthocyanin derivatives and aliphatic glucosinolates in A. thaliana.This pattern of metabolite production aligns well with the results reported earlier by Kusano et al. (2011), who measured the metabolite content in A. thaliana seedlings grown in Petri dishes, and also with the more recent measurements reported by Job et al. (2022).This trend of metabolite accumulation measured in Col-0 contrasted significantly with what reported in B. rapa, where almost all metabolites, with the only exception of the indolic glucosinolate 4-hydroxyindolyl-3-methyl glucosinolate (4MOI3M), registered a temporal increase (Fig. 6; Fig. S1).Indolic glucosinolates, and in particular 1-methoxy-indolyl-3-methyl glucosinolate (1MOI3M), indolyl-3-methyl glucosinolate (I3M), and 4MOI3M, decreased also in B. napus upon UV-B treatment.As for the accumulation of anthocyanin derivatives, these compounds revealed a decreased accumulation upon UV-B treatment in all species, with the exception of B. napus (Fig. S1).We next focused attention on the accumulation of phenylacylated flavonoids, as the additional phenolic ring deriving from the sinapoyl decoration enhances the spectral absorption and consequently the UV-B shielding properties of glycosylated flavonoids (Tohge et al. 2016).In A. thaliana, all four classes of sinapoyl derivatives, which includes sinapoyl-glucoside (SinGlc), sinapoyl-malate (SinMal), di-sinapoyl-glucoside (Sin-Glc-Sin) and sinapoylated anthocyanin (A11, cyanidin 3 O-(malonyl) glucoside) revealed an increased accumulation with the exposure to UV-B (Fig. 6).In all the remaining Brassicaceae, only sinapoyl-glucoside derivatives revealed an increased accumulation in all species, while Sin-Glc-Sin and A11 were not detected.
In conclusion, all Brassicaceae species displayed the ability to mount a response to UV-B light, which includes transcriptional activation of the phenylpropanoid pathway, followed by the regulation of primary metabolism, ultimately culminating with synthesis and accumulation of specialized metabolites for the conferral of increased protection.As the analysis of primary metabolites revealed a widespread increase in Phe in all species, differences that we measured in the content of specific specialized metabolites could be attributed to species-specific activation of enzymes involved in the chemical decoration of flavonoid molecules.

Conclusion
In this study, we examined the metabolic and transcriptional responses to UV-B radiation of four Brassicaceae species, including the model plant A. thaliana ecotype Col-0 and compared the results with responses in B. napus, B. oleracea, and B. rapa.Our analysis revealed a complete reprogramming of the pathway of the central metabolism, leading to a variation of metabolite abundance already visible after 2 h of UV-B treatment.Reprogramming of the central metabolism became even more evident after 10 h of exposure, revealing species-specific variation of metabolite abundance.Nonetheless, the immediate precursors of the flavonoid pathway and flavonoid decorations, such as the amino acid Phe, increased in all species.Species-specific variation was also observed in the content of specialized metabolites although sinapoyl glucose derivatives revealed a typical increase in all Brassicaceae species.Nonetheless, the analysis of SCPL transcript abundance displayed constitutive expression in all Brassicaceae and downregulation in response to the treatment with UV-B irradiation.Our study also suggested that the downregulation of SCPL expression is independent of the HY5 signaling cascade, which instead showed the conventional induction in all the Brassicaceae as previously reported for A. thaliana.

Plant material and growth
Seeds of A. thaliana ecotype Col-0, B. napus, B. oleracea, and B. rapa were surface-sterilized in 30% (v:v) bleach in water for 5 min, followed by a 5-min rinse with sterilized water.Sterilized seeds were sown on Petri dishes filled with half-strength Murashige and Skoog salts (Murashige and Skoog, 1962) containing 1% (w/v) sucrose and 1% agar (w/v) and vernalized at 4 ∘ C in the dark for 3 d.At the end of the vernalization period, the seeds were left to germinate for 10 d in a growth chamber set with the following conditions: light intensity, 180 μmol m −2 s −1 ; photoperiod, 8/16 h:light/dark; temperature, 22 ∘ Cand humidity, 75%.Uniformly germinated seedlings were transferred to pots and grown under long-day conditions (photoperiod, 16/8 h:light/dark) for the full duration of the experiment.UV-B radiation (0.8 μmol m −2 s −1 ; 315 nm) was applied to 2-week-old (transcriptional analysis) and 4-week-old (transcriptional and metabolomic analyses) seedlings starting at 9:00 AM when the plants had already received 3 h of visible photosynthetic radiation.Plant material was harvested at different time points as specified in the text using time 0 as a negative control.Measurements of metabolites were conducted in triplicate.

Analysis of metabolites via GC-MS and LC-MS
Plant material was ground to fine powder using a ball mill (Tesch, Haan, Germany) at liquid N 2 temperature and stored at −80 ∘ C until metabolite extraction.Primary and specialized metabolites were extracted from 50 mg of leaf powder with 0.7 ml of methanol containing ribitol (2 mg .l −1 ) and isovitexin (5 mg .l −1 ) as internal standards.A phase separation with chloroform was used to remove chlorophylls before separating each sample into two aliquots for the analysis of primary and specialized metabolites, respectively.Primary metabolites were derivatized with ethoxyamine hydrochloride in pyridine and measured via GC-MS following the procedure described by Lisec et al. (2006) and Alseekh et al. (2021).Specialized metabolites were separated and measured via LC-MS following the protocols previously described by Tohge and Fernie (2010) and Perez de Souza et al. (2021).

Phylogenetic analysis of SCPL genes
Orthologs of A. thaliana SCPL genes in the Brassicaceae species were identified as previously described (Tohge et al. 2016).Sequences of SCPL genes were aligned with MUSCLE and used to build the phylogenetic tree in MEGA 7.0 using the maximum likelihood method.Short genes (CRU_004G02050, TP4G01870, TP4G01860, TP4G01990 and TP4G02020) were excluded from this analysis.

Gene expression analysis by qRT-PCR
RNA extraction from plant tissues was performed using TRIzol reagent (Ther-moFisher, Carlsbad, CA, USA).Digestion with DNase and cDNA synthesis were performed with Maxima First Strand cDNA Synthesis Kit with dsDNase (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania).The integrity of the RNA was verified on 1% (w/v) agarose gels, and the concentration was measured using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).A final volume of 5 μl was used to run qRT-PCR reactions (Caldana et al. 2007).Data analysis was performed using SDS software version 2.4 (Applied Biosystem, Foster City, CA, USA) normalized to the expression of UBQ10 housekeeping gene.The oligonucleotides used for the qRT-PCR analyses are provided in Supplementary Table S3.Three or six replicates were utilized to measure gene expression.

Fig. 1
Fig. 1 The transcriptional activation of HY5 in response to UV-B exposure.(A) A schematic representation of the duration of the UV-B treatment (thick purple line) and time points of sample harvest (dots on the thin blue line).Gray and white squares represent dark and light photoperiods, respectively.(B) The abundance of HY5 transcripts normalized by the expression of UBQ10 at different time points during the UV-B treatment.Each bar represents the means ± Standard Error (SE) (n = 3).Bars marked with an asterisk are significantly different at P < 0.05.LD, length of the day.

Fig. 3
Fig. 3 The abundance of SCPL transcripts in response to UV-B exposure.The abundance of SCPL transcripts normalized by the expression of UBQ10 at different time points during the UV-B treatment.Each bar represents the means ± Standard Error (SE) (n = 3).Bars marked with an asterisk are significantly different at P < 0.05.

Fig. 4
Fig. 4 Metabolites of the central pathway.(A) A schematic representation of the duration of the UV-B treatment (thick purple line) and time points of sample harvest (dots on the orange line).Gray and white squares represent dark and light photoperiods, respectively.(B) A schematic representation of the central metabolic pathway.Heatmaps along the pathway represent log 2 fold changes of metabolite abundance measured in leaves at time point 2 and 10 h versus time 0. Metabolites marked with an asterisk are significantly different at P < 0.05.AAA, aromatic amino acid; AT, A. thaliana; BCAA, branched amino acids; BN, B. napus; BO, B. oleracea; BR, B. rapa; F6P, fructose-6-phosphate; GABA, γ-aminobutyric acid; GLS, glucosinolate; G6P, glucose-6-phosphate; OAS, O-acetyl-serine; Sin, sinapoyl; LD, long-day; TCA, tricarboxylic acid cycle.

Fig. 5
Fig. 5 The transcriptional activation of the phenylpropanoid pathway in response to UV-B exposure.Transcript abundance normalized by the expression of UBQ10 after 6 and 8 h of exposure to UV-B light.Each bar represents the means ± Standard Error (SE) (n = 6).Bars marked with an asterisk are significantly different from the untreated control at P < 0.05.